Molecular Imaging Identifies Regions with Microthromboemboli During Primary Angioplasty in Acute Coronary Thrombosis

Animal Preparation and Experimental Protocol

In the past, models of acute myocardial infarction (AMI) have generally used coronary ligation to produce coronary occlusion (5–9). In the clinical setting, however, sudden coronary occlusion is most often associated with coronary thrombosis. We have developed a model of acute coronary thrombosis in dogs (10) that we used for this study, which was approved by the Animal Research Committee at the University of Virginia and conformed to the position of the American Heart Association on Research Animal Use. Twenty adult mongrel dogs were anesthetized with 30 mg·kg⁻¹ of sodium pentobarbital and mechanically ventilated with a respirator pump. An 8-French catheter sheath was placed in the left carotid artery for coronary angiography and PTCA. Similar catheters were placed in both femoral arteries for duplicate reference sample withdrawal during radiolabeled microsphere injection for measuring regional myocardial blood flow (MBF), and one of them was connected to a multichannel recorder for arterial pressure monitoring. Catheters were placed in both femoral veins for administration of fluids and infusion of microbubbles. A heated mattress was used to maintain the core body temperature at 38°C.

A left lateral thoracotomy was performed and the heart was suspended in a pericardial cradle. A 6-French catheter was placed in the left atrium for injection of radiolabeled microspheres. MBF measurements and coronary angiography were performed at baseline (Fig. 1). The mid and distal portions of the left anterior descending coronary artery (LAD) were then dissected free from surrounding tissues. In 15 dogs, a PTCA balloon (3.0-mm diameter, 9.0-mm length) was advanced into the LAD over a guide wire. Umbilical tape was tightened around the distal LAD, and the mid-LAD was subjected to external injury using forceps. Balloon inflation was started at 6–8 atmospheric pressure 20 mm proximal to the distal ligation site for 50–200 min (mean, 110 min). The different occlusion times were used to create thrombi of different sizes.

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FIGURE 1.

Time-line diagram of sequence of experimental procedures.

In 5 control dogs, the LAD was occluded for approximately 150 min by tightening an umbilical tape around it without producing a coronary thrombus. These dogs were used since microthrombi can occur within the ischemic myocardium even in the absence of coronary thrombosis because of local glycoprotein IIb/IIIa and α_vβ₃ activation (11–13).

Just before recannalization, myocardial contrast echocardiography (MCE) was performed to measure the risk area (RA) and radiolabeled microspheres were injected for measurement of MBF. ^99mTc-DMP-444 was administrated intravenously for in vivo labeling of the coronary thrombus (Fig. 1). Thereafter, the LAD ligation was reversed and PTCA was performed in the LAD injury dogs if an occlusive thrombus was seen on coronary angiography. MBF as well as hemodynamic data were measured at both 30 and 60 min after successful recanalization in all dogs. We chose these 2 times after reperfusion because we have previously shown that MTE can spontaneously dissolve between these times after reperfusion (10) and we wanted to study the effect of the magnitude of MTE on the spontaneous resolution of no reflow. At the end of the experiment, sutures were placed on the myocardium at the level of the ultrasound transducer to identify the MCE imaging plane postmortem. The dogs were then killed with a solution of pentobarbital and potassium and the heart was removed. The slice corresponding to the MCE plane was then cut for ^99mTc autoradiography, IS measurement, and radiolabeled microsphere-derived MBF determination (Fig. 1). Using a single heart slice we were able to register all datasets accurately.

Thrombus Imaging

In vivo labeling of the coronary thrombus was achieved by intravenous administration of 37 MBq·kg⁻¹ of ^99mTc-labeled DMP-444 (DuPont Pharmaceuticals). This agent binds to the glycoprotein IIb/IIIa platelet receptor, allowing imaging of the thrombus (3). It is a hydrazinenicotinamide-functionalized compound (cyclo[d-Val-NmeArg-Gly-Asp-Mamb(5(6-(6-hydrazinonicotinamido)hexamide))] [HYNICitide]) labeled with ^99mTc using tricine and a water-soluble phosphine as coligands. Its 50% inhibitory concentration for the IIb/IIIa receptor is 11 nmol/L·L⁻¹ (4).

At the end of the experiment, the heart slice corresponding to the MCE image was placed directly on the collimator of a γ-camera (Orbiter 37; Siemens). A high-resolution collimator with a 140-keV, 20% window centered around the photopeak of ^99mTc with a 128 × 128 matrix was used. Image quantification was performed on a nuclear medicine computer (ICON; Siemens) (14).

For quantification of regional myocardial ^99mTc activity, regions of interest were drawn over the background-subtracted RA and the normal contralateral wall, and the average activity in these regions were measured. A count ratio was computed by dividing the average counts per pixel in the RA by the average counts per pixel in the contralateral wall. The hot spot was defined as the region with more than the mean + 2 SD of the activity in the normal myocardium (as defined by MCE) and was planimetered and expressed as a percentage of the left ventricular (LV) short-axis area.

MBF Measurements

MBF was measured with left atrial injections of 2·10⁶, 11-μm radiolabeled microspheres (DuPont Medical Products) suspended in 4 mL of 0.9% saline and 0.01% Tween-80 (15). The microspheres, their energy windows, and half-lives are as follows: ¹¹³Sn, 340–440 keV, 115 d; ¹⁰³Ru, 450–550 keV, 39 d; and ⁴⁶Sc, 842–1,300 keV, 84 d. None of these isotopes has kiloelectron volts in the range of that of ^99mTc. Duplicate reference blood samples (10 mL each) were withdrawn from the femoral arteries over 130 s with a constant-rate withdrawal pump (model 944; Harvard Apparatus). After autoradiography, the LV short-axis slice corresponding to the MCE image was cut into 16 wedge-shaped pieces, excluding the papillary muscles, and each piece was further divided into epicardial, mid-myocardial, and endocardial segments. The tissue and reference blood samples were counted 1 wk later in a well counter with a multichannel analyzer to allow for complete decay of ^99mTc. Corrections were made for activity spilling from one energy window to another with a custom-designed program (14).

MBF to each segment was calculated using the equation Qm = (Cm·Qr)·Cr, where Qm is blood flow to the myocardial segment (mL·min⁻¹), Cm is tissue counts, Qr is rate of arterial sample withdrawal (mL·min⁻¹), and Cr is arterial reference sample counts (15). The absolute MBF (mL·min⁻¹·g⁻¹) to each of the 48 pieces was calculated as the quotient of the flows and the weight of the segment.

MBF in each segment within the LAD was also represented with a parametric image with color coding to display the magnitude of MBF. This custom-designed program uses colors ranging from brown (low flow) to bright yellow (high flow) (16). No reflow was defined when the ratio of endocardial MBF in the center of the RA and MBF in the normal myocardium (defined by MCE)⁻¹ (MBF ratio) was <0.25 at both 30 and 60 min after recanalization. This criterion was used because it has been previously shown that no reflow and necrosis are closely correlated and that they are seen only when MBF < 25% of normal (6–9,17,18).

RA Measurement

RA was measured during coronary occlusion using MCE. Intermittent harmonic imaging was performed using a phased-array system (Power Vision 6000; Toshiba) with a transducer that transmits ultrasound at a mean frequency of 2.1 MHz and receives 4.2 MHz. Six end-systolic images were obtained at a pulsing interval of 10 cardiac cycles during a continuous infusion of 2 mL of Definity (Bristol Myers Squibb Medical Imaging) diluted in 23 mL of 0.9% saline.

Images were aligned using computer cross-correlation as previously described (16–18). Color coding was applied to background-subtracted images to visually enhance regional differences in myocardial contrast enhancement (16–18). The RA was planimetered by an observer who was unaware of the region with no contrast enhancement and was expressed as a percentage of the LV short-axis slice as previously described (16–18). We and others have validated this method of RA assessment against postmortem techniques (17–20).

IS Determination

At the end of the experiment, the heart was excised, and a 1-cm slice corresponding to the MCE imaging plane was immersed in a solution of 1.3% 2,3,5-triphenyltetrazolium chloride (SigmaCorp.) and 0.2 mol/L Sorensen’s buffer in distilled water (pH 7.4) at 37°C for 20 min, followed by fixation in 10% formalin (21). The apical and basal sides of the stained specimen were imaged with a digital scanning camera. The regions with unstained myocardium on both aspects of the slice was measured by planimetry and averaged. Infarct size was determined as a percentage of the LV short-axis area.

Statistical Methods

Results are expressed as mean ± 1 SD. Data were compared using the Mann–Whitney U test and correlations were assessed using by the Pearson correlation coefficient. Receiver operating characteristic (ROC) curves were generated to determine the best cutoff for predicting AMI using the ^99mTc count ratio. All findings were considered significant at P < 0.05 (2-sided).

Critique of Methods

Since we were using a ^99mTc-labeled compound for assessing the degree of MTE, we could not use a similar compound for the assessment of RA. It is for this reason that we used MCE to define RA. This in vivo technique has been well validated for this purpose (17–20) and has a higher spatial resolution for defining RA than radiolabeled microsphere–derived MBF.

Platelet activation or plugging can occur locally within the RA even in the absence of MTE. Furthermore, α_vβ₃ activity in platelets as well as the vascular endothelium is also upregulated during ischemia, and an agent that binds to the IIb/IIIa receptor may also cross-react with the α_vβ₃ receptor (11–13). It is for this reason that we used control dogs undergoing coronary ligation without thrombus production. Though these dogs also showed the presence of activated IIb/IIIa in the RA, the magnitude and spatial extent of the signal was much less than that of the dogs with vascular injury. As expected, the activity was the greatest in dogs who demonstrated a thrombus on coronary angiography.

We performed quantitative ^99mTc autoradiography by directly placing the heart slice on the collimator of a γ-camera rather than using film. In this manner, our count ratios were accurate. The minimal loss in spatial resolution was not a major concern because of the large differences in the spatial extent of MTE between the groups. We used a single heart slice over which the ultrasound transducer was fixed during MCE and which was then used for autoradiography, determining IS, and MBF measurements.

A limitation of the study was that we did not use histopathology to confirm the presence of microthrombi in areas with DMP-444 activity. However, this has been validated by others previously (3,4). Another limitation is that we did not study a model of atherosclerosis in which a residual coronary stenosis could have been present, potentially requiring stent placement.

Clinical Implications

These results have an important bearing on the understanding of the relationship between no reflow and necrosis because of the controversy regarding the temporal relationship between microvascular abnormalities and myocyte injury. The conventional wisdom is that the microvascular phenomenon is a consequence of cell death (22). This belief is based on animal models of coronary ligation and not thrombosis. Studies using MCE in patients with AMI have reported that the low-reflow phenomenon can be partially reversed by the administration of verapamil and nicorandil and that this reversal is associated with improved regional function and outcome (23,24). Taken together with the results of our study, it appears that in the setting of coronary thrombosis, part of the no reflow may be caused by MTE and may precede necrosis unless MTE is treated.

Thus, our results imply that measures aimed at reducing the thrombus burden may reduce MTE and IS. These measures could include mechanical means of preventing thrombus from migrating distally and pharmacologic measures directed toward attenuating platelet activation. For instance, a recent study showed that intracoronary thrombectomy with the X-sizer catheter system improved epicardial flow and accelerated ST-segment resolution in patients with acute coronary syndrome (25). Improved TIMI grade flow has also been reported in patients with AMI receiving IIb/IIIa receptor antagonists (26). In an experimental model of coronary thrombosis and PTCA, we have recently shown that the combined blockade of IIb/IIIa and α_vβ₃ reduced IS substantially (27). Molecular imaging of MTE may play a role in clinical trials designed to study such approaches in patients with acute coronary syndromes.